α-Synuclein liquid condensates fuel fibrillar α-synuclein growth

α-Synuclein (α-Syn) aggregation into fibrils with prion-like features is intimately associated with Lewy pathology and various synucleinopathies. Emerging studies suggest that α-Syn could form liquid condensates through phase separation. The role of these condensates in aggregation and disease remains elusive and the interplay between α-Syn fibrils and α-Syn condensates remains unexplored, possibly due to difficulties in triggering the formation of α-Syn condensates in cells. To address this gap, we developed an assay allowing the controlled assembly/disassembly of α-Syn condensates in cells and studied them upon exposure to preformed α-Syn fibrillar polymorphs. Fibrils triggered the evolution of liquid α-Syn condensates into solid-like structures displaying growing needle-like extensions and exhibiting pathological amyloid hallmarks. No such changes were elicited on α-Syn that did not undergo phase separation. We, therefore, propose a model where α-Syn within condensates fuels exogenous fibrillar seeds growth, thus speeding up the prion-like propagation of pathogenic aggregates.

Figs. S1 to S6 Table S1 Legends for movies S1 to S8

Other Supplementary Material for this manuscript includes the following:
Movies S1 to S8  Bars,2µm. (D) Epi-�uorescence microscopy images of HeLa cells expessing the α-Syn-emGFP fusion. Bar, 10µm for whole cell images and 2µm for zoom-ins. (E) FRAP curves displaying the �uorescence recovery of α-Syn-enriched condensates 24H (blue, 10 cells) and 72H (red, 15 cells) after cell transfection with the α-Syn_emGFP_5Fm scaffold. Dotted line in bold represents the mean and the error bar represents the standard deviation. Aquisitions were made every second. Pre-bleaching �uorescence intensity was set at 100 and the post-bleach �uorescence was normalized at 0 for all experiments. (F) Time-lapse imaging of the dissolution of 72H-old control (Upper row) and α-Syn (Lower row) condensates upon the addition of FK506. Bar, 10μm. . Abnormally-shaped condensates occur only from α-Syn condensates exposed to fibrils (A)Quanti�cation of the percentage of cells with condensates expressing the two different phenotypes: "Regular"-blue; "Abnormal" -red. as a function of time and concentration of preformed �brils. HeLa cells expressing α-Syn-emGFP-5Fm (for 24 hours, 48 hours and 72 hours) were exposed to increasing concentration of preformed �brils (0.01 nM, 0.1 nM and 0.5 nM). The graph is the sum of N=2 experiments. (B) HeLa cells expressing control (left) and α-Syn (right) condensates and exposed for 72 hours to preformed α-Syn oligomers (0.5 nM). (C) HeLa cells expressing α-Syn-emGFP-5Fm scaffold (green) and exposed to preformed α-Syn �brils (red) for 72 hours in presence of FK506. Nuclei (cyan) were stained with Hoechst. Bars, 10 µm (D) FRAP analysis of control condensates from cells exposed (dark blue, 8 cells) or not (light blue, 15 cells) to �brils. Pre-bleaching �uorescence intensity was set at 100 and the post-bleach �uorescence was normalized at 0 for all experiments. An acquisition was made every second. Error bars correspond to the standard deviation. (E) HeLa cells expressing α-Syn condensates (green) and exposed to preformed α-Syn �brils (red) for 72 hours after incubation with FK506 for 1 hour. Nuclei (cyan) were stained with Hoechst. Bars, 10 μm for full-cell images and 2 μm for zoom-ins. (F) Live confocal microscopy frames depicting the FK506-driven dissolution patterns of 24 hours-old (upper pannel) and 48 hours-old (lower pannel) α-syn condensates (green) from HeLa cells exposed to �brils (red). Different timepoints are shown, with 0 s being pinpointed at the moment when FK506 was added. Bars, 10 µm for whole cell frames and 2 µm for zoom-ins.

Fig. S5. α-Syn condensates are perturbed by preformed ribbons
Representative epi�uorescence images of HeLa cells exposed to preformed α-Syn ribbons (red) and expressing control (A) and α-Syn (B) condensates (green). Nuclei (cyan) were stained with Hoechst. Bars, 10 µm for full-cell images and 2 µm for zoom-ins. (C) Quanti�cation of the percentage of cells with condensates expressing the two different phenotypes: "regular"-blue; "abnormal" -red. The graph is the sum of N=3 experiments. (D) Quanti�cation of the Pearson coefficient calculated for the correlation between the �uorescence intensities of the ribbon and condensate channels in microscopy images that were cropped to match the shape of the cell. Each point represents a cell. The graph is the sum of N=3 experiments. Differences between conditions with control and α-Syn condensates were statistically signi�cant (p-values < 10-7 using a Student's t-test). a.u. stands for arbitrary unit. (E) Live confocal microscopy frames depicting HeLa cells expressing α-Syn condensates (green) changing morphology upon exposure to ribbons (red). Bars, 10 µm for full-cell images and 2 µm for zoom-ins. Representative epi�uorescence images of SHSY-5Y cells infected with preformed α-Syn �brils (red) and expressing control (A) and α-Syn (B) condensates (green). Nuclei (cyan) were stained with Hoechst. AmyTracker staining on SHSY-5Y cells expressing α-Syn condensates after 72 hours incubation with �bres. Bars, 10 μm for full-cell images and 2 μm for zoom-ins. (C) FRAP analysis of abnormally-shaped condensates. The graph comprises the average of 10 different experiments for the condition with �brils and 7 different experiments for the condition without �birls . Pre-bleaching �uorescence intensity was set at 100% and the post-bleach �uorescence was normalized at 0 for all experiments. An acquisition was made every second. Error bars correspond to the standard deviation.

Supplementary movies
Movie S1: Time-lapse confocal microscopy of the formation of α-Syn condensates. Acquisitions were taken every 5 minutes.
Movie S2: Time-lapse confocal microscopy of the formation of control condensates. Acquisitions were taken every 5 minutes.
Movie S3: Time-lapse epifluorescence microscopy of the dissolution of 24H-old α-Syn condensates. FK506 was added 30 seconds in. Acquisitions were taken every 10 seconds.
Movie S4: Time-lapse epifluorescence microscopy of the dissolution of 72H-old α-Syn condensates. FK506 was added 30 seconds in. Acquisitions were taken every 10 seconds.
Movie S5: Time-lapse confocal microscopy of the evolution of α-Syn condensates (green) from "regular" to "abnormal" phenotype in presence of fibres (red). Acquisitions were started 48 hours after cell exposure to fibrils and images were taken every 5 minutes.
Movie S6: Time-lapse epifluorescence microscopy of the dissolution of 72H-old α-Syn condensates (green) with fibres (red). FK506 was added 30 seconds in. Acquisitions were taken every 10 seconds.
Movie S7: Time-lapse confocal microscopy of the growth of α-Syn condensates (green) in presence of fibres (red). Acquisitions were taken every 5 minutes.
Movie S8: Time-lapse confocal microscopy of the evolution of α-Syn condensates (green) from "regular" to "abnormal" phenotype in presence of ribbons (red). Acquisitions were started 48 hours after cell exposure to ribbons and images were taken every 5 minutes